Project Summary The unfolded protein response (UPR), a well-known adaptive mechanism for cells to maintain ER homeostasis, has been implicated in the pathogenesis of a variety of human diseases. Genetic deletion of the UPR signaling components can significantly influence the disease progression in mouse models for diabetes, hepatic steatosis, IBD, ALS and other diseases. We have recently performed a synthetic biology screen to interrogate the mammalian UPR pathway. From this screen, we have identified RtcB as a long sought RNA ligase that catalyzes unconventional XBP-1 splicing during ER stress. RtcB is a multifunctional RNA ligase that also acts in tRNA splicing. Genetic rescue of RtcB knockout cells suggests that RtcB-mediated ligation of XBP1 and tRNA may occur at different subcellular compartments. We will therefore determine subcellular compartment-specific RtcB function. By generating Trpt1 and RtcB double knockout cells and performing genetic rescue, we further demonstrate that a second biochemically distinct RNA ligation pathway cooperates with RtcB in mammalian cells to regulate XBP1 and tRNA splicing. Therefore, we will examine the role of other putative components in the RNA ligation pathway. Lastly we will identify unknown RNA ligases of the new ligation pathway by a genetic suppressor screen using RtcB knockout cells. By revealing the complete molecular repertoire of RNA ligation pathways, we will hopefully gain new insights and uncover new therapeutic targets to modulate the mammalian UPR.